17. Antibiotics Discovery: From Hit to Lead and Lead to Candidate Drug
Antibiotics: Hit to Lead and Lead to Candidate Drug (H2L and L2CD)
Lecture Outline
Characterizing a Hit
Identifying and de-risking liabilities
Lead declaration
Candidate Drug Criteria
Note: The lecture focuses on the discovery of antibiotics targeting WHO Priority Gram-negative pathogens:
Carbapenem-resistant Enterobacteriaceae (E. coli, K. pneumoniae)
P. aeruginosa and A. baumannii
However, the same principles apply to discovery and development regardless of target species.
Defining a Hit Compound
There is no universally accepted definition of a ‘Hit’. However, the definitions given here are commonly accepted in Big Pharma.
A ‘Hit’ must be deemed sufficiently worthwhile to explore and develop (spending time & money). This involves assessing its potential for clinical translation and commercial viability.
Biochemical Hit
Structure known: The chemical structure of the compound must be elucidated and confirmed.
Preliminary SAR (structure-activity relationship): Initial understanding of how structural modifications affect activity. This helps in guiding further chemical optimization.
Amenable to chemical alteration / production: The compound should be synthetically tractable, allowing for modifications to improve its properties. Scalable synthesis is crucial for further development.
Interacts with target (protein): The compound must bind to the intended biological target.
Inhibits target (protein) activity: Binding should lead to a measurable reduction in the target's activity.
Whole-Cell Antibacterial Hit
Biochemical Hit attributes PLUS in addition:
Inhibits bacterial growth (of key pathogens): Demonstrates antibacterial activity against relevant bacterial strains.
MIC ≤ 16 µg/ml versus key pathogens (initial value): The minimum inhibitory concentration (MIC) should be at or below this level, indicating reasonable potency.
Activity against resistant strains: The compound should retain activity against strains exhibiting common resistance mechanisms. This is critical for addressing unmet clinical needs.
Activity not due to non-specific activity (detergent-like): The antibacterial effect must be specific to the biological target, not due to general cytotoxic effects.
Lead Compound
Good antibacterial & safety profile AND efficacy in mouse models. A lead compound should show promising results in preclinical studies, supporting its potential for further development.
Hit to Lead Screening Assays
Physiochemical properties (Mol Wgt, pKA, solubility, LogD, etc): Molecular weight, acid dissociation constant, solubility, distribution coefficient, etc. These parameters affect drug behavior in biological systems.
Antibacterial activities (MIC): Minimum inhibitory concentration against relevant bacterial strains.
Resistance (FoR): Frequency of resistance, which measures the likelihood of bacteria developing resistance to the compound.
Target inhibition (biochemical activity): Biochemical assays to confirm target inhibition.
Cytotoxicity: Assessment of the compound's toxicity to mammalian cells.
ADME properties: Absorption, distribution, metabolism, and excretion properties, which determine how the drug behaves in the body.
Human target liabilities: Potential interactions with human targets that could cause adverse effects.
PK and Formulation: Pharmacokinetics (PK) and formulation studies to assess drug exposure and delivery.
Tolerability and efficacy in in vivo models: Animal studies to evaluate the drug's tolerability and efficacy.
The following slides will be a tour through these important assays made during H2L and L2CD.
Physiochemical Properties of the Compound
Acid Dissociation Constant (pKa)
pH at which 50% of (part of) a drug is ionized.
pKa influences lipophilicity, solubility, protein binding, and permeability, which in turn directly affects pharmacokinetic (PK) characteristics such as absorption, distribution, metabolism, and excretion (ADME). Understanding pKa is crucial for predicting a drug's behavior in vivo.
Examples of clinical antibiotic pKa values
Antibiotics with multiple pKa values (contain more than one ionizable group)
Zwitterions
Contains an equal number of positively- and negatively-charged functional groups.
Importance of zwitterions: Studies of β-lactam antibiotic uptake through OmpF/C porins of E. coli reveals that Zwitterionic molecules are preferred over negatively charged compounds by the general porins of Enterobacteria. Zwitterionic properties can enhance bacterial uptake.
Many approved antibiotics are zwitterions
Zwitterionic Forms of Antibiotics
Ciprofloxacin (fluoroquinolone) diffusion through the inner membrane: Ciprofloxacin exists in zwitterionic form, which aids its diffusion.
Ciprofloxacin in the zwitterionic form stacks on the edge of the IM (Ciprofloxacin diffuses in water as concerted stacks). These stacks dissolve as they enter the membrane. Individual molecules then diffuse through the membrane as neutral CIP monomers due to intermolecular transfer of protons.
Neutral and zwitterionic CIP coexist at physiological pH, with the zwitterionic form being predominant.
Solubility
Kinetic solubility: The solubility of the fastest dissolving species. Can be done from a stock solution in DMSO. Rapid & cheap - used to rank compounds (<20 µM = poor; 20-80 µM = moderate; >80 µM = good). Use: Develop structure-solubility relationships. This is a quick screen for identifying compounds with poor solubility.
Thermodynamic solubility: By addition of aqueous solvent directly to solid crystalline material. Gives the equilibrium solubility of all species. Time and resource-consuming one compound at a time. Use: Guides formulation development. Provides accurate data for formulation design.
IMPORTANT: If a compound has low solubility, then other assay results may not be reliable. Low solubility can lead to inaccurate assay results.
Lipophilicity/Hydrophilicity: LogD
The distribution coefficient (LogD) between two immiscible solvent phases- A lipophilic organic phase and a polar aqueous phase - octanol/water.
LogD at pH 7.4 (LogD7.4) gives an indication of the lipophilicity of a drug molecule at the pH of blood plasma. This is a key parameter for predicting drug distribution in the body.
For ionizable compounds (remember pKa), LogD is altered by pH because the distribution of charged and uncharged forms would change, and the uncharged form is more hydrophobic.
CMC (chemistry, manufacturing, and controls) drug database:- All drugs: mean calculated LogD (at pH 7.4) = 1.6
Gram-positive antibiotics mean cLogD = −0.2 (high aqueous solubility)
Gram-negative antibiotics mean cLogD = −2.8 (high aqueous solubility)
Remember, this is a log scale – these differences are large
cLogD = calculated LogD Critical information – can modify LogD by chemistry
Antibiotics vs. Other Drugs: LogD
Antibiotics are very different to other drugs – lower LogD (pH 7.4) values
PP = Antibiotics acting in the Periplasmic space – e.g., beta-lactams
CP = Antibiotics acting in the Cytoplasmic space – e.g., fluoroquinolones
Antibacterial Activity of Compounds (MIC)
Primary Panel
4 key Gram-negative pathogens: Assess the spectrum of Gram-negative activity. Assess efflux and entry liabilities- E. coli (Wild-type, Efflux-defective, Hyperporinated)
K. pneumoniae (Wild-type)
P. aeruginosa (Wild-type, Efflux-defective)
A. baumannii (Wild-type)
1 Gram-positive: Assess broader spectrum of activity- S. aureus (Wild-type)
8 strain panel: Fits on a 96-well microtiter plate (8 strains x 10 drug concentrations, plus positive and negative controls)
Compound concentration range: 64 – 32 – 16 – 8 – 4 – 2 – 1 – 0.5 – 0.25 – 0.125
MIC assays conducted according to approved CLSI or EUCAST protocols
MIC <8 preferred (ultimately for most programmes Lead will have a MIC < 2)
Secondary Panel
MIC assay against a panel of clinical MDR isolates (10-20 strains)
Include examples of common clinical resistance types- ESBL, CarbapenemR, FluoroquinoloneR, TetracyclineR, ColistinR, etc, etc.
Examples should be appropriate to the species covered by the Hit
Essential that the Hit is unaffected by existing resistance mechanisms
No shift in MIC (≤ 2-fold shift acceptable) with known MDR mechanisms
MIC in the Presence of Human Serum
MIC assay against primary panel WT strains with 50% human serum added
Quick and easy assay to perform
Ideally, no shift in MIC (a shift suggests plasma protein binding that could reduce bioavailability in vivo). A shift suggests increased plasma protein binding, which can reduce bioavailability.
MIC90
Measure MIC of recent clinical isolates (50 - >100) of each species of interest for the compound class
MIC90 is the lowest compound concentration at which ≥ 90% of the isolates are inhibited
Identify whether clinical isolates have a higher MIC than primary panel strains
Is there any pre-existing resistance in the natural/clinical bacterial population?- Unimodal population: Good outcome
Bimodal population (pre-existing resistance): Bad outcome – terminate project
Time-Kill Assay
Time to kill as a function of exposure to different compound concentrations
Gives information on:- Whether compound is bacteriostatic or bactericidal
How quickly it kills
Whether resistant mutants arise in the culture
Samples taken at 24 hours and MIC measured- Are they resistant mutants?
TK information is used in: PKPD modelling to guide dosing in animals (Lena Friberg)
Resistance Development: Frequency of Resistance (FoR)
Aim: Is spontaneous resistance a liability?
Measure agar MIC for compounds
Plate cultures on 4 x MIC and 8 x MIC
Incubate 24, 48 h
Count colonies (calculate frequency)
Pick an streak colonies on selective agar
If they regrow then measure MIC
Resistant mutants have increased MIC
Cultures can be serially passaged in antibiotic to select multiple mutations to increased resistance
Relative fitness of mutants can be tested by growth rate (or growth competition)
Bad outcome:- FoR >
Fold-increase in MIC large
Fitness of mutants high
Good outcome:- FoR <
Fold-increase in MIC small
Fitness of mutants low
Resistance Mechanisms (WGS, genetics)
Aim: Identify all mechanisms of resistance
Target mutations causing resistance (also confirms target)
Efflux as a source of resistance (complements MIC)
Cell wall/membrane mutants as source of resistance
Amplifications as source of resistance
Information on target mutations could help medicinal chemistry to design compounds that overcome resistance
Useful to know if resistance mechanisms are the same between different species
Mutations might affect drug target in one species BUT drug efflux in a different species
May help drug identify target
Target Binding or Inhibition Assay (SPR)
A simple general binding assay is SPR
Binding measured as a change in resonance units (RUs) on the biosensor surface.
Develop a bespoke assay for a project
Measure IC50 of target-binding interaction or target enzyme inhibition
Aim: show that improvements in MIC correlate with target inhibition
SPR – surface plasma resonance (Biacore) assays determine the affinity and binding kinetics of a ligand for its receptor. SPR provides quantitative data on binding affinity.
Measures real-time binding association and dissociation rates using Surface Plasmon Resonance (SPR).
Cytotoxicity
In vitro cytotoxicity (often in HepG2 cells): Assessment of toxicity in liver cells.
Viability of HepG2 cells can be measured in several different types of assay.- Measure cellular ATP levels
Measure fluorescent dyes crossing membrane
Half-maximal inhibitory concentrations (IC50) are calculated.
For antibiotics, IC50 >100 µM is usually preferred
Haemolysis of red blood cells (human)- Measure release of haemoglobin
Microtiter plate assay – read OD (include positive & negative controls)
Acceptable: <1% haemolysis at 100 µM after 1h at 37°C
Mutagenicity/Carcinogenicity
Ames assay- Assesses whether the compound causes an increase in mutation frequency – an indication that it is a mutagen and therefore potentially a carcinogen.
Uses Salmonella his mutants as the assay system - with and without the addition of liver extract
Mutagenic compound = BAD
Micronucleus test- Assesses whether the compound causes chromosome damage (visualized as the presence of micronuclei in peripheral blood erythrocytes) – an indication that a compound is a carcinogen.
Required outcome: negative on both assays
ADME and Related Assays
Plasma Protein Binding (human, mouse, rat)
Measure compound binding to plasma proteins.
Affects PK and PD
Examples: 18% ampicillin; 97% dicloxacillin; novobiocin 99%.
Plasma protein binding is important because:- Only the non-protein-bound fraction of an antibiotic is microbiologically active.
Only the non-protein-bound fraction of a drug in plasma can penetrate into the extravascular space.
Penetration into the extravascular space is highly important for antimicrobial therapy, as the majority of bacterial infections occur in the interstitial fluid of tissues or in body fluids other than blood.
Preferred outcome: < 90%, with no interspecies variation
Microsomal Stability Assay (mouse, rat, dog, monkey, human)
Liver microsomes are a heterogeneous set of vesicles 20-200nm in diameter that are formed from the endoplasmic reticulum when cells are disrupted.
Liver microsomes contain a wide variety of drug-metabolizing enzymes and are commonly used to support in vitro metabolism studies
Metabolic stability is defined as the percentage of the parent compound lost over time, assessed in the presence of liver microsomes. Metabolic stability affects drug clearance.
The purpose of the microsomal stability assay is to calculate the rate of intrinsic drug clearance (and whether it differs with species).
Metabolite profiling- Use Mass Spec to identify major metabolites
Can apply chemistry to make the Hit more stable (or less stable) as required
Preferred outcome: t1/2 > 30 minutes
Caco-2 Permeability & Efflux Assay
Caco-2 is a cell line derived from a human colon carcinoma.
Resembles the enterocytes lining the small intestine
Caco-2 permeability assay measures the rate of flux of a compound across Caco-2 cell monolayers.
Test compound concentrations are quantified using a calibration curve following analysis by LC-MS/MS, and the apparent permeability coefficient (Papp) and efflux ratio of the compound across the monolayer are calculated.
The efflux ratio is used as an indicator of active efflux.
The data generated can predict in vivo absorption of drugs, suitability for oral dosing, intestinal permeability, and provides information on drug efflux from human cells.
Compound is added at a concentration of 10 µM and applied to the apical and/or basolateral kits’ compartment for 2 hours, to evaluate unidirectional or bidirectional transport between compartments.
Required: > cm/s if oral TPP
Human Target Liabilities
hERG (the human Ether-à-go-go-Related Gene)- The alpha subunit of a potassium ion channel (hERG).
The hERG ion channel helps coordinate the heart's beating. hERG inhibition is a critical safety concern.
Inhibition by drugs can result in a potentially fatal disorder called long QT syndrome (a fatal irregularity of the heartbeat).
This has made hERG inhibition an important anti-target that must be avoided during drug development.
NaV1.5- NaV1.5 is a sodium ion channel subunit primarily in cardiac muscle
Mediates the fast influx of Na+-ions across the cell membrane, resulting in the fast depolarization phase of the cardiac action potential.
Summary:
It is critical to determine early in Hit development whether compounds have human ion channel liabilities
Tested channels include hERG, Nav 1.5, and Cav 1.2 (+ others)
All associated with cardiac problems.
Preferred outcome: IC50 > 100 µM
Cytochrome P450 3A4 (CYP3A4)
An important enzyme in the body, mainly found in the liver and in the intestine.
It oxidizes small foreign organic molecules including toxins and drugs, so that they can be removed from the body.
Importantly, some drugs inhibit CYP3A4, which can affect the metabolism of a variety of other drugs, increasing their bioavailability. In some cases, this can have fatal consequences. Careful attention to CYP3A4 is vital in drug development.
Some drugs can induce CYP3A4, thus affecting the turnover of other drugs in the body.
Multiple CYP enzymes will be tested eventually before clinical trials
CYP inhibition preferred outcome: IC50 > 10 µM
CYP induction preferred outcome: No or limited induction
Bacterial Selectivity: Mitochondrial Toxicity
Need to set up assays to monitor toxicity if the drug target has a human homolog
For example:- aminoglycosides are potentially toxic to human mitochondrial ribosomes.
If developing a new aminoglycoside one must assay this toxicity.
CEREP Assay
Eurofins-CEREP is a major company that profiles interactions between development compounds and a large panel of human targets (including ion channels, discussed earlier).
If target interactions occur then each must be individually assessed as a potential liability
Required result: No target inhibited more than 50% at 30 µM in a panel of human enzymes/receptors/transporters.
Formulation and Tolerability
The chemical and physical properties of the compound are determined in order to develop a safe, effective, and stable dosage formulation for Pre-Clinical studies
Cyclodextrins are cyclic oligosaccharides used for the improvement of water solubility and bioavailability of drugs. Often used in formulations.
Mouse tolerability Study- Determine the MTD (maximum tolerated dose) before beginning an efficacy study
Mouse Pharmacokinetics (PK)
Aim: Determine the compound exposure (concentration) as a function of time after treatment – in a variety of different tissues.
Delivery routes: PO, IV, SC, etc
Provides useful information before beginning efficacy studies
Provides useful information on possible toxicity issues
Drug concentration / time in different tissues
Route of excretion and metabolism
Efficacy Study: Mouse Infection
Aim: Does the compound reduce the bacterial infection? Is there evidence of a dose response?
Positive result: ≥ 1 log reduction in cfu relative to SOT
Lead Declaration: Required Information
Understanding the Compound
Structure of the best compound
Patent space (or freedom to operate)
Calculated properties of the compound (MW, HBD/HBA, etc., etc)
Physicochemical properties (logD at pH 7.4, pKa, chemical stability, solubility)
Understanding the Activity
Target overview (MoA, biochemical potency, selectivity, structure of target if available)
Synthesis (SAR, steps in synthesis, overall yield, lowest yield step)
ADME (Stability in microsomes and hepatocytes, CYP3A4 inhibition / induction, Caco-2 permeability, etc, etc)
Liability screening (Cytotoxicity HepG2, hERG, Nav 1.5, Genotoxicity, Haemolysis, in vitro pharmacology – CEREP)
In vitro antibacterial profile (MICs, MIC90, MIC-MDR, cidal/static, FoR, Cross-resistance, Specificity on target)
In vivo pharmacology (Tolerability in mice, efficacy model - formulation, administration, dose)
In vivo / in vitro PK (Plasma stability, protein binding, mouse PK)
Small molecule drug: You are now several years into the process, and have probably made > 400 compounds
Lead to Candidate Drug Phase
A Candidate Drug is regarded as ready (safe and efficacious) to go into human clinical trials
H2L assays (MIC, cytotox, etc) continue to be made on new L2CD analogues
Optimisability- No more than 3-4 significant issues after H2L stage
Optimisation plan- Make an action plan based on the identified issues/liabilities
Chemistry to solve issues by making and testing new analogues
Identify PK/PD Drivers of in vivo Efficacy
See Lena Friberg's lectures for more details, especially on modeling and allometry
Candidate Criteria: Preclinical PK Profile
Acceptable PK in at least two species (rodent & non-rodent)
Sufficient exposure to allow tox studies
Low blood clearance (< 60% liver blood flow)
Rodent toxicology:- By the intended route of administration (7-day tox study)
>10-fold margin for AUC and Cmax for efficacy versus AUC and Cmax at 7-day NOAEL
Beyond the standard Hit to Lead assays
NOAEL = No-Observed-Adverse-Effect Level
Rodent & Non-Rodent Toxicology
Establish a maximum tolerated dose (MTD) in the rat model (then in non-rodent models – dog, then minipig)
Followed this by a repeat dose phase of 3, 7, 10 or 14 days
Maximum Tolerated Dose (MTD)
Dose Range Finding (DRF)- High dose – MTD from MTD/DRF studies
Low dose (NOAEL)
Mid-dose (geometric mean of High and Low)
Aim is to establish an intended therapeutic dose for FTIH
Candidate Criteria
Patentability:- Acceptable IP protection and FTO for Candidate
Synthetic Route:- Suitable to deliver large batches (kilogram scale)
Acceptable cost of goods (COG) At predicted dose and targeted indications
Purity and characterization: ≥98% preferred Well characterized (e.g., by NMR, LC/MS, MS/MS, etc)
Genotoxic risk assessment: Completed with plans to control in candidate formulation Both in manufacture and stability for clinical studies
FTO = Freedom To Operate
COG = Cost of Goods
Candidate Criteria: Stable Version & Solubility
Stable version available: Salt form available for development of IV formulation Crystalline, non-hygroscopic preferred No conversions at 20 – 70% relative humidity at 25°C Water uptake <2% at 75% relative humidity
Solubility: >5 mg/mL in vehicle for oral or i.v. formulation
Pharmaceutical Development Prepare application for Phase I clinical trials